1. Abstract
Whitney Heuer, Andrea Gonzalez, LuAnn Scott, Holly A. Wichman, and Christine E. Parent
Department of Biological Sciences, University of Idaho, Moscow, ID 83844-3051, heue2741@vandals.uidaho.edu
Viral infections represent a serious health concern for human
populations. Viruses have the ability to mutate, evolve in response
to new environments, and may change in virulence over time.
Experimental models can help shed light on how viruses change
and evolve over time. We are using Drosophila melanogaster to
develop a model to study and evaluate the course of viral infection
and innate immune responses. Specifically, we orally infected
experimental populations of D. melanogaster with Drosophila X
virus (DXV) to mimic natural infection. After passage 3 and
passage 4, RT-PCR was perform using dead and living flies from
the treatments to determine if DXV was still present. We were able
to detect DXV within on line of treatment. Development of this
system will help to establish a system for the study of evolution of
viruses within host populations.
Background
Characteristics of Drosophila melanogaster
• Innate immune response (no acquired response)
• Easy to handle and care for
• Large populations readily acquired
• Fly-specific viruses available and characterized
Drosophila X Virus (DXV)
• Family Birnaviridae
• Double stranded RNA genome with 2 segments
• Capsid arranged in T=13 lattice
• Icosahedral with hexagonal outline
• Diameter 58-60 nm
• Unenveloped
• Causes anoxia sensitivity in D. melanogaster
Experimental Design
Oral Infection- Day 0
Day (-4) – Day (6)
PCR Passage 4
• PCR detected virus within the diluted virus passage, replicate 4
(R4).
• Further treatments have been done since, but PCR results were
negative for all of the replicates.
Conclusion
• Natural transmission can occur between generations infected with
Drosophila X virus by feeding of smashed flies.
• In the near future, we will conduct further experiments with different
treatments to see if the virus sustains longer with different
techniques of collection, infection, and transfer of flies.
ACKNOWLEDGEMENTS
• This work was funded by the National Institutes of Health
under grant No. P20GM104420.
• We thank Tanya Miura, Ashley DeAguero, and the Fly-Virus
group at the University of Idaho for assistance with this work.
• http://jvi.asm.org/ for fly images
Serial Passaging of Drosophila X Virus in Drosophila melanogaster
Objective
Detect DXV in Drosophila melanogaster by serial passaging using
infected flies from previous treatments.
• Harvest flies day -3
• Infect day 0
• Collect dead day 1-
day 6
• Collect alive flies day
6
Passage 0
• Harvest flies day -3
• Infect day 0 with
smashed flies
• Collect dead day 1-6
• Collect alive flies day
6
Passage 1 • Harvest flies day -3
• Infect day 0 with
smashed flies
• Collect dead day 1-6
• Collect alive flies day
6
Passage 2
• Harvest flies day -3
• Infect day 0 with
smashed flies
• Collect dead day 1-6
• Collect alive flies day
6
Passage 3 • Harvest flies day -3
• Infect day 0 with
smashed flies
• Collect dead day 1-6
• Collect alive flies day
6
Passage 4
• At 8am, transfer 20 harvested flies
from day -3 into empty vials.
• Starve for 4 hours
• At 12 pm, infect flies with
DXV.
• Allow to feed for 6 hours
• At 6 pm,
transfer
flies to
food vials.
Concentrated Oral Inoculation (P0-R1-3)
Combine:
- 50 µL 25% sterile sucrose
- 450 µL DXV stock
Dilute Oral Inoculation (P0-R4)
- 50 µL 25% sterile sucrose
- 225 µL DXV stock
- 225 µL S2 media
Infect Passage 1- Passage 4
Combine:
- 20 flies per replicate (dead and
alive)
- Smash flies with100 µL of 50 mM
Tris-Cl, pH 7.5 for 1’30”
- Combine 100 µL of fly extract with
10 µL 25% sucrose
Day -4
Dump
generation
to be
harvested
Day -3
Collect and
harvest 20
flies per
replicate
Day 0
Infection
Day 1-5
Collect
dead flies
Day 6
/Day -1
Collect dead
and alive flies
to infect next
passage
R1 R2 R3 R4 R1 R2 R3 R4 +